Project Summary The Vaccine Branch FACS (Flow Cytometry) Core Facility has been created to meet an unmet need on the Bethesda Campus of NCI for a Flow Cytometry Core Facility with BL2/BL3 containment necessary to safely sort live cells infected with retroviruses, such as HIV/SIV, and other infectious agents known or suspected in blood and tissue samples. The facility has a multicolor cell sorter currently in use for standard and infectious sorting. In addition, this facility has analysis capabilities from five multicolor flow cytometry instruments that were donated to the facility from three Investigators in the CCR, and through IATAP funding. Laboratory space renovations were completed in May 2008, and the donated flow cytometers were relocated to the facility in June 2008. The facility purchased a 4 laser high end analyzer and a 3 laser high speed cell sorter. These were purchased at the beginning of FY09 and installed in Q2 FY09. The facility currently maintains two investigator-run 2 laser 4 color analog flow cytometers, one investigator-run 2 laser 6 color digital flow cytometer, one investigator-run 3 laser 12 color digital high end flow cytometer, and one investigator-run 4 laser 18 color high end digital flow cytometer. The 3 laser digital high end flow cytometer has undergone extensive hardware and software upgrades to add additional fluorescent and sample processing abilities, and both of the 2 laser analog flow cytometers have been upgraded to a new computer system. The facility has a 3 laser 12 color digital cell sorter equipped with containment for use as an infectious cell sorter. The sorter is fully operational and has been used for infectious sorting as of Q3 FY09. The Facility Manager has trained 4 investigators to operate the cell sorter for their individual projects. The facility currently supports more than 70 NCI/CCR Investigators from the Vaccine Branch, the Laboratory of Receptor Biology and Gene Expression, the Laboratory of Cellular Oncology, and the Laboratory of Cancer Biology and Genetics. In addition to NCI investigators, the Facility Manager is working on a collaboration with an investigator from the FDA. Experiments currently conducted in the facility involve multi-parameter single cell analysis of cells from human, non-human primate and mouse primary sources. Additionally analysis is also conducted on in vitro cultured cells. Reagents for these experiments include monoclonal and polyclonal antibodies with fluorescent labels and fluorescent dyes used for staining a variety of cellular components. Current applications being supported by the facility include multi-color phenotypic analysis, cell cycle analysis, apoptosis analysis, proliferation analysis with BrdU and CFSE, intracellular cytokine analysis, rare event analysis and calcium flux analysis. Currently supported projects include, but are not limited to, immune monitoring for HIV, SIV, HTLV-1 and HTLV-2 vaccine studies on non-human primates, and monitoring the results of gene expression on cell proliferation and apoptosis.